scholarly journals BPGAP1 Interacts with Cortactin and Facilitates Its Translocation to Cell Periphery for Enhanced Cell Migration

2004 ◽  
Vol 15 (6) ◽  
pp. 2873-2883 ◽  
Author(s):  
Bee Leng Lua ◽  
Boon Chuan Low
Keyword(s):  
Cell Migration ◽  
Control Cell ◽  
Actin Binding ◽  
Ionization Mass ◽  
Cell Periphery ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Cell Dynamics

Rho GTPases control cell dynamics during growth and development. They are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). Many GAPs exist with various protein modules, the functions of which largely remain unknown. We recently cloned and identified BPGAP1 as a novel RhoGAP that coordinately regulates pseudopodia and cell migration via the interplay of its BNIP-2 and Cdc42GAP homology, RhoGAP, and the proline-rich domains. To further elucidate the molecular mechanism underlying cell dynamics control by BPGAP1, we used protein precipitations and matrix-assisted laser desorption/ionization mass spectrometry and identified cortactin, a cortical actin binding protein as a novel partner of BPGAP1 both in vitro and in vivo. Progressive deletion studies confirmed that cortactin interacted directly and constitutively with the proline-rich motif 182-PPPRPPLP-189 of BPGAP1 via its Src homology 3 domain. Together, they colocalized to periphery and enhanced cell migration. Furthermore, substitution of prolines at 184 and 186 with alanines abolished their interaction. Consequently, this BPGAP1 mutant failed to facilitate translocation of cortactin to the periphery, and no enhanced cell migration was observed. These results provide the first evidence that a RhoGAP functionally interacts with cortactin and represents a novel determinant in the regulation of cell dynamics.

Filling the GAPs in cell dynamics control: BPGAP1 promotes cortactin translocation to the cell periphery for enhanced cell migration

2004 ◽  
Vol 32 (6) ◽  
pp. 1110-1112 ◽  
Author(s):  
B.L. Lua ◽  
B.C. Low
Keyword(s):  
Cell Migration ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Small Gtpases ◽  
Cell Periphery ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Cell Dynamics ◽  
Cellular Division

Cells undergo dynamic changes in morphology or motility during cellular division and proliferation, differentiation, neuronal pathfinding, wound healing, apoptosis, host defense and organ development. These processes are controlled by signalling events relayed through cascades of protein interactions leading to the establishment and maintenance of cytoskeletal networks of microtubules and actin. Various regulators, including the Rho small GTPases (guanine nucleotide triphosphatases), serve as master switches to fine-tune the amplitude, duration as well as the integration of such circuitry responses. Rho GTPases are activated by guanine nucleotide-exchange factors and inactivated by GAPs (GTPase-activating proteins). Although normally down-regulating signalling pathways by catalysing their GTPase activity, many GAPs exist with various protein modules, the functions of which still largely remain unknown. BPGAP1 is a novel RhoGAP that co-ordinately regulates pseudopodia and cell migration through the interplay of its BNIP-2 and Cdc42GAP homology domains serving as a homophilic/heterophilic interaction device, an enzymic RhoGAP domain that inactivates RhoA and a proline-rich region that binds the Src homology-3 domain of cortactin. Both proteins co-localize to cell periphery and enhance cell migration. As a molecular scaffold in cortical actin assembly and organization, cortactin and its interaction with small GTPases, GAPs and tyrosine kinases seems set to provide further insights to the multiplicity and complexity of cell dynamics control. Elucidating how these processes might be individually or co-ordinately regulated through cortactin remains an exciting future challenge.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals CRMP2 as a Candidate Target to Interfere with Lung Cancer Cell Migration

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1533
Author(s):  
Xabier Morales ◽  
Rafael Peláez ◽  
Saray Garasa ◽  
Carlos Ortiz de Solórzano ◽  
Ana Rouzaut
Keyword(s):  
Cell Migration ◽  
Lung Adenocarcinoma ◽  
Cancer Cell ◽  
Adaptor Protein ◽  
Mechanistic Explanation ◽  
In Vitro Assays ◽  
Cancer Cell Migration ◽  
Cortical Actin

Collapsin response mediator protein 2 (CRMP2) is an adaptor protein that adds tubulin dimers to the growing tip of a microtubule. First described in neurons, it is now considered a ubiquitous protein that intervenes in processes such as cytoskeletal remodeling, synaptic connection and trafficking of voltage channels. Mounting evidence supports that CRMP2 plays an essential role in neuropathology and, more recently, in cancer. We have previously described a positive correlation between nuclear phosphorylation of CRMP2 and poor prognosis in lung adenocarcinoma patients. In this work, we studied whether this cytoskeleton molding protein is involved in cancer cell migration. To this aim, we evaluated CRMP2 phosphorylation and localization in the extending lamella of lung adenocarcinoma migrating cells using in vitro assays and in vivo confocal microscopy. We demonstrated that constitutive phosphorylation of CRMP2 impaired lamella formation, cell adhesion and oriented migration. In search of a mechanistic explanation of this phenomenon, we discovered that CRMP2 Ser522 phospho-mimetic mutants display unstable tubulin polymers, unable to bind EB1 plus-Tip protein and the cortical actin adaptor IQGAP1. In addition, integrin recycling is defective and invasive structures are less evident in these mutants. Significantly, mouse xenograft tumors of NSCLC expressing CRMP2 phosphorylation mimetic mutants grew significantly less than wild-type tumors. Given the recent development of small molecule inhibitors of CRMP2 phosphorylation to treat neurodegenerative diseases, our results open the door for their use in cancer treatment.

scholarly journals A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

2004 ◽  
Vol 15 (11) ◽  
pp. 5158-5171 ◽  
Author(s):  
Pieta K. Mattila ◽  
Omar Quintero-Monzon ◽  
Jamie Kugler ◽  
James B. Moseley ◽  
Steven C. Almo ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
C Terminus ◽  
Actin Binding Domain ◽  
Affinity Interaction

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 μM) compared with ATP-G-actin (Kd 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved β-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

scholarly journals Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology.

1993 ◽  
Vol 120 (2) ◽  
pp. 503-512 ◽  
Author(s):  
F Widmer ◽  
P Caroni
Keyword(s):  
Phosphorylation Site ◽  
Growth Cones ◽  
Cell Cortex ◽  
Membrane Association ◽  
Major Protein ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Cortical Cytoskeleton

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane-association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP-43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin-containing cortical cytoskeleton in a regulatable manner.

scholarly journals In Vivo Importance of Actin Nucleotide Exchange Catalyzed by Profilin

2000 ◽  
Vol 150 (4) ◽  
pp. 895-904 ◽  
Author(s):  
Amy K. Wolven ◽  
Lisa D. Belmont ◽  
Nicole M. Mahoney ◽  
Steven C. Almo ◽  
David G. Drubin
Keyword(s):  
Point Mutations ◽  
Fluid Phase ◽  
Intrinsic Rate ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
Cytoskeleton Organization

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP2 and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin–cofilin generated during filament disassembly.

SYK regulates B-cell migration by phosphorylation of the F-actin interacting protein SWAP-70

Blood ◽  
2011 ◽  
Vol 117 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Glen Pearce ◽  
Tatsiana Audzevich ◽  
Rolf Jessberger
Keyword(s):  
Cell Migration ◽  
B Cells ◽  
B Cell ◽  
Cell Polarization ◽  
Actin Binding ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Interacting Protein

Abstract B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.

scholarly journals Tropomyosin isoforms bias actin track selection by vertebrate myosin Va

2016 ◽  
Vol 27 (19) ◽  
pp. 2889-2897 ◽  
Author(s):  
Maria Sckolnick ◽  
Elena B. Krementsova ◽  
David M. Warshaw ◽  
Kathleen M. Trybus
Keyword(s):  
Full Length ◽  
Actin Binding ◽  
Myosin Isoforms ◽  
Cortical Actin ◽  
Actin Binding Domain ◽  
Myosin Motor Domain ◽  
Myosin Va ◽  
Temporal Localization

Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin–Tpm3.1 and actin–Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin–Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an “actin–Tpm code” that allows for spatial and temporal sorting of actomyosin function in the cell.

scholarly journals DENND2B activates Rab13 at the leading edge of migrating cells and promotes metastatic behavior

2015 ◽  
Vol 208 (5) ◽  
pp. 629-648 ◽  
Author(s):  
Maria S. Ioannou ◽  
Emily S. Bell ◽  
Martine Girard ◽  
Mathilde Chaineau ◽  
Jason N.R. Hamlin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cancer Progression ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Leading Edge ◽  
Cell Periphery ◽  
Nucleotide Exchange ◽  
Migrating Cells

The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Förster resonance energy transfer–based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell’s leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers.

scholarly journals PLC-γ1 and Rac1 Coregulate EGF-Induced Cytoskeleton Remodeling and Cell Migration

2009 ◽  
Vol 23 (6) ◽  
pp. 901-913 ◽  
Author(s):  
Siwei Li ◽  
Qian Wang ◽  
Yi Wang ◽  
Xinmei Chen ◽  
Zhixiang Wang
Keyword(s):  
Cell Migration ◽  
Rho Gtpases ◽  
Sh3 Domain ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cytoskeleton Remodeling ◽  
Src Homology

Abstract It is well established that epidermal growth factor (EGF) induces the cytoskeleton reorganization and cell migration through two major signaling cascades: phospholipase C-γ1 (PLC-γ1) and Rho GTPases. However, little is known about the cross talk between PLC-γ1 and Rho GTPases. Here we showed that PLC-γ1 forms a complex with Rac1 in response to EGF. This interaction is direct and mediated by PLC-γ1 Src homology 3 (SH3) domain and Rac1 106PNTP109 motif. This interaction is critical for EGF-induced Rac1 activation in vivo, and PLC-γ1 SH3 domain is actually a potent and specific Rac1 guanine nucleotide exchange factor in vitro. We have also demonstrated that the interaction between PLC-γ1 SH3 domain and Rac1 play a significant role in EGF-induced F-actin formation and cell migration. We conclude that PLC-γ1 and Rac1 coregulate EGF-induced cell cytoskeleton remodeling and cell migration by a direct functional interaction.

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